RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing

Abstract

We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3′gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2 kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3′gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.

Non-GM Genome Editing Approaches in Crops

“…Sequencing of the targeted genomic region detected no edits but addition of a cassette containing the viral RNA silencing suppressor, p19, to the gRNA vector produced gene editing in systemic tissues ( Zhang et al, 2020 ). These results are in line with recent research indicating that RNA silencing suppressors can increase genome editing efficiency ( Mao et al, 2018 ; Zhang et al, 2020 ; Chiong et al, 2021 ; Mao et al, 2021 ). Unfortunately, the authors did not attempt to regenerate plants from systemically infected tissue or test heritable editing in the progeny of infected plants and previous studies have failed to obtain edited seeds ( Zhang et al, 2020 ).…”

“…A non-systemic PSV expression vector based on the tobacco mosaic virus was also developed which expressed both Cas9 and gRNA for gene editing in the presence of p19 , a viral suppressor of RNA silencing. However, no attempt was made to regenerate gene edited plants ( Chiong et al, 2021 ).…”

Functional Characterization of Genes Coding for Novel β-D-Glucosidases Involved in the Initial Step of Secoiridoid Glucosides Catabolism in Centaurium erythraea Rafn

“…Transient expression of recombinant proteins in plants using tobacco mosaic virus (TMV) based viral vectors have been well documented ( Chiong et al, 2021 ). TMV-based overexpression pJL-TRBO vector can be used for the insertion of a target gene by agroinfiltration into plants.…”

VIGE: virus-induced genome editing for improving abiotic and biotic stress traits in plants

“…Although most other CRISPR/Cas9 virus systems are intended to generate heritable mutations, the focus of this TMV system is to provide transient editing technology complementary to established VIGS methods (Cody et al 2017). A follow-up study further optimized the system using RNA interference suppressors and simultaneously delivering Cas9 and gRNAs from a single TMV construct to eliminate the need for transgenic plants or co-delivery of the components from separate constructs (Chiong et al 2021). Though editing efficiency was lower when using a single construct compared to co-delivery, it was nonetheless possible to obtain ~7% editing efficiency in N. benthamiana even with the large insert load of about 4.2 kb (Chiong et al 2021).…”